Comparative analysis of 16S rRNA signature sequences of the genus Idiomarina and Idiomarina woesei sp. nov., a novel marine bacterium isolated from the Andaman Sea

A Gram-negative, short rod, aerobic bacterium, designated W11^T, was isolated from seawater. Heterotrophic growth was observed at 10–45 °C and pH 6–10. Optimal growth was observed at 30–37 °C and pH 7–9. It can grow in the presence of 0.5–12% NaCl (w/v), and the optimal NaCl required for growth was 5–6%. 16S rRNA gene sequence similarity revealed that strain W11^T clustered within the radiation of the genus Idiomarina and showed 99.24% similarity with Idiomarina donghaiensis JCM 15533^T, 97.64% with Idiomarina marina JCM 15083^T, 97.37% with Idiomarina tainanensis JCM 15084^T and 97.16% with Idiomarina maritima JCM 15534^T. DNA–DNA similarities between strains W11^T with other closely related strains were below 70%. Polar lipids included a phosphatidylgylycerol, a diphosphatidylglycerol, a phosphatidylethanolamine, an unidentified phosopholipid, two unidentified aminolipids and two unidentified lipids. DNA G + C content was 41.2 ± 0.1 mol%. Major fatty acids were iso-C_15:0, iso-C_17:0, iso-C_17:1ω9c, C_16:0, iso-C_11:0 3OH and C_16:1 ω7c/C_16:1 ω7c. The isoprenoid ubiquinone was Q8. On the basis of the present polyphasic taxonomic study, strain W11^T is considered to represent a novel species of the genus Idiomarina, for which the name Idiomarina woesei sp. nov. is proposed. The type strain is W11^T (= DSM 27808^T = JCM 19499^T = LMG 27903^T).     

Vibrio panuliri sp. nov., a marine bacterium isolated from spiny lobster,Panulirus penicillatus and transfer of Vibrio ponticus from Scophthalmi clade to the newly proposed Ponticus clade

A novel marine bacterium, strain LBS2^T was isolated from eggs carried on pleopods of the spiny lobster collected from Andaman Sea. Heterotrophic growth occurred at 1–7% NaCl. 16S rRNA gene sequence similarity revealed the strain LBS2^T belonged to the genus Vibrio and showed above 97% similarity with eight type strains of the genus Vibrio. Multilocus analysis based on ftsZ, gapA, gyrB, mreB, pyrH recA, rpoA, and topA revealed LBS2^T formed a separate cluster with Vibrio ponticus DSM 16217^T with 89.8% multilocus gene sequence similarity. However, strain LBS2^T is distantly related with other members of the Scophthalmi clade in terms of 16S rRNA signatures, phenotypic variations and multilocus gene sequence similarity, for which we propose LBS2^T belongs to a new clade i.e. Ponticus clade with V. ponticus DSM 16217^T as the representative type strain of the clade. DNA–DNA homologies between strain LBS2^T and closely related strains were well below 70%. DNA G + C content was 45.3 mol%. On the basis of our polyphasic study, strain LBS2^T represents a novel species of the genus Vibrio, for which the name Vibrio panuliri sp. nov. is proposed. The type strain is LBS2^T (= JCM 19500^T = DSM 27724^T = LMG 27902^T).     

Novosphingobium pokkalii sp nov,a novel rhizosphere-associated bacterium with plant beneficial properties isolated from saline-tolerant pokkali rice

Pokkali rice varieties are known for their saline tolerance when specifically grown in coastal saline affected agri-fields of southern Kerala. These fields are prone to seawater intrusion. During characterization of phytobeneficial rhizobacteria from this pokkali rice, L3E4^T was isolated. This strain showed some plant growth-promoting functions (production of indole acetic acid (IAA), acetoin, and siderophore), biofilm formation and capacity to use a wide range of plant-derived organic compounds. In planta assay under axenic conditions showed a positive effect of L3E4^T on pokkali rice growth; importantly, it was able to attach and colonize pokkali rice roots in the presence of natural seawater, a key adaptation required for survival in pokkali rice fields. Phylogenetic analysis using 16S rRNA, recA, and gyrB gene sequences showed that strain L3E4^T belongs to the genus Novosphingobium, with Novosphingobium capsulatum GIFU 11526^T and Novosphingobium rhizosphaerae JM.1^T being the nearest phylogenetic relatives. In addition, DNA–DNA hybridization analysis and phenotypic traits established that this strain belongs to a novel Novosphingobium species, for which we propose the name Novosphingobium pokkalii sp. nov. The type strain is represented by strain L3E4^T (=MTCC 12357^T = KCTC 42224^T).     

Thermoregulation, pacing and fluid balance during mass participation distance running in a warm and humid environment

Deep body temperature (T _c), pacing strategy and fluid balance were investigated during a 21-km road race in a warm and humid environment. Thirty-one males (age 25.3 ± 3.2 years; maximal oxygen uptake 59.1 ± 4.2 ml kg⁻¹ min⁻¹) volunteered for this study. Continuous T _c responses were obtained in 25 runners. Research stations at approximately 3-km intervals permitted accurate assessment of split times and fluid intake. Environmental conditions averaged 26.4°C dry bulb temperature and 81% relative humidity. Peak T _c was 39.8 ± 0.5 (38.5–40.7) °C with 24 runners achieving T _c > 39.0°C, 17 runners ≥39.5°C, and 10 runners ≥40.0°C. In 12 runners attaining peak T _c ≥ 39.8°C, running speed did not differ significantly when T _c was below or above this threshold (208 ± 15 cf. 205 ± 24 m min⁻¹; P = 0.532). Running velocity was the main significant predictor variable of ∆T _c at 21 km (R ² = 0.42, P < 0.001) and was the main discriminating variable between hyperthermic (T _c ≥ 39.8°C) and normothermic runners (T _c < 39.8°C) up to 11.8 km. A reverse J-shaped pacing profile characterised by a marked reduction in running speed after 6.9 km and evidence of an end-spurt in 16 runners was observed. Variables relating to fluid balance were not associated with any T _c parameters or pacing. We conclude that hyperthermia, defined by a deep body temperature greater than 39.5°C, is common in trained individuals undertaking outdoor distance running in environmental heat, without evidence of fatigue or heat illness.     

HPA and SAS responses to increasing core temperature during uncompensable exertional heat stress in trained and untrained males

Increases in core temperature (T _c) augment stress hormones and neurotransmitters; however, the effect of different T _c tolerated with varying fitness levels during uncompensable exertional heat stress (EHS) is unclear. The purpose was to examine the hypothalamic–pituitary–adrenal (HPA) axis and sympathetic-adrenomedullary system (SAS) responses during uncompensable EHS in trained (TR) versus untrained (UT) males. Twelve TR and 11 UT ( [(V)\dot]\textO_2\textpeak = 70 ±2 \dot{V}{\text{O}}_{{2{\text{peak}}}} = 70 \pm 2 and 50 ± 1 mL kg of lean body mass⁻¹ min⁻¹) walked on a treadmill to exhaustion (EXH) in 40°C (dry), dressed in protective clothing. PRE and 0.5°C T _c increments from 38.0–40.0°C/EXH venous blood was obtained. Cortisol responded to absolute thermal strain, increasing throughout EHS and independent of fitness. Adrenocorticotropic Hormone, Norepinephrine, and Dehydroepiandrosterone–Sulphate responded to relative thermal strain with similar EXH values, despite higher T _c tolerated for TR (39.7°C) than UT (39.0°C). Epinephrine, Growth Hormone (GH), and Aldosterone increased initially, with a plateau above 38.5°C T _c. Findings demonstrate the complexity of the HPA axis, SAS, and T _c relationship, with the stress pathways responding largely to relative thermal strain, although some hormones exhibited a clamping response likely as a protective mechanism. For the TR, evidence existed for a reduced pituitary sensitivity to glucocorticoids and the amplified GH may have contributed to their greater T _c tolerated.     

Peripheral markers of central fatigue in trained and untrained during uncompensable heat stress

The development of fatigue is more pronounced in the heat than thermoneutral environments; however, it is unclear whether biomarkers of central fatigue are consistent with the higher core temperature (T _c) tolerated by endurance trained (TR) versus untrained (UT) during exertional heat stress (EHS). The purpose of this study was to examine the indicators of central fatigue during EHS in TR versus UT. Twelve TR and 11 UT males (mean ± SE [(V)\dot]\textO _{2 \textpeak} \dot{V}{\text{O}}_{{ 2 {\text{peak}}}}  = 70 ± 2 and 50 ± 1 mL kg LBM⁻¹ min⁻¹, respectively) walked on a treadmill to exhaustion (EXH) in 40°C (dry) wearing protective clothing. Venous blood was obtained at PRE and 0.5°C T _c increments from 38 to 40°C/EXH. Free tryptophan (f-TRP) decreased dramatically at 39.5°C for the TR. Branch chain amino acids decreased with T _c and were greater for UT than TR at EXH. Tyrosine and phenylalanine remained unchanged. Serum S100β was undetectable (<5 pg mL⁻¹). Albumin was greater for the UT from PRE to 39.0°C and at EXH. Prolactin (PRL) responded to relative thermal strain with similar EXH values despite higher T _c tolerated for TR (39.7 ± 0.09°C) than UT (39.0 ± 0.09°C). The high EXH PRL values for both groups support its use as a biomarker of the serotonin and dopamine interplay within the brain during the development of central fatigue.     

Sulfoacidibacillus ferrooxidans,gen. nov., sp. nov., Sulfoacidibacillus thermotolerans,gen. nov., sp. nov., and Ferroacidibacillus organovorans,gen. nov., sp. nov.: Extremely acidophilic chemolitho-heterotrophic Firmicutes

Ten strains of extremely acidophilic bacteria, isolated from different environments form a distinct monophyletic clade within the phylum Firmicutes. Comparison of complete genomes of the proposed type strains confirm that they comprise two genera (proposed names Sulfoacidibacillus and Ferroacidibacillus), and at least three species (Sulfoacidibacillus ferrooxidans, Sulfoacidibacillus thermotolerans and Ferroacidibacillus organovorans). The bacterial strains share some physiological traits, including catalysing the dissimilatory oxidation and reduction of iron, and in being obligately heterotrophic. Both species of Sulfoacidibacillus are also able to oxidise elemental sulfur and tetrathionate. Both S. ferrooxidans and Ferroacidibacillus spp. are mesophilic, while S. thermotolerans isolates are moderate thermophiles. The isolates display different degrees of acid-tolerance: Ferroacidibacillus spp. are the most acid-sensitive while the type strain of S. ferrooxidans grows at pH 0.9. MK7 was detected as the sole menaquinone present in all three nominated type strains, and their peptidoglycans all contain meso-2,6 diaminopimelic acid type A1γ. The chromosomal DNA of the strains examined contain between 44 and 52 mol% G + C. The nominated type strains of the new species are S. ferrooxidans S⁰AB^T (= DSM 105355^T = JCM 33225^T); S. thermotolerans Y002^T (= ATCC TSD-104^T = JCM 31946^T); F. organovorans SLC66^T (= ATCC TSD-103^T = JCM 31945^T).     

Description of Klebsiella africanensis sp. nov., Klebsiella variicola subsp. tropicalensis subsp. nov. and Klebsiella variicola subsp. variicola subsp. nov.

The bacterial pathogen Klebsiella pneumoniae comprises several phylogenetic groups (Kp1 to Kp7), two of which (Kp5 and Kp7) have no taxonomic status. Here we show that group Kp5 is closely related to Klebsiella variicola (Kp3), with an average nucleotide identity (ANI) of 96.4%, and that group Kp7 has an ANI of 94.7% with Kp1 (K. pneumoniae sensu stricto). Biochemical characteristics and chromosomal beta-lactamase genes also distinguish groups Kp5 and Kp7 from other Klebsiella taxa. We propose the names Klebsiella africanensis for Kp7 (type strain, 200023^T = CIP 111653^T) and K. variicola subsp. tropicalensis for Kp5 (type strain, 1266^T = CIP 111654^T).     

Is the circadian core temperature rhythm of juvenile rats due to a periodic blockade of thermoregulatory thermogenesis?

Previous studies have demonstrated an endogenous circadian rhythm of core temperature (T _c) in suckling-age rat pups. Our aim was to establish whether the low and irregular T _c at the beginning of the light phase is caused by a temporary blockade of thermoregulatory thermogenesis. We therefore isolated and artificially fed 10-day-old pups for 30 h at five ambient temperatures (T _a), ranging from thermoneutrality to severe cold loading, while continuously recording T _c and metabolic rate (MR). During the maximum phase of the T _c rhythm MR increased linearily with decreasing T _a — to as much as 260% of the daily mean MR at thermoneutrality (TNMR) — so that T _c decreased less than 1°C with increasing cold load. During the minimum phase, the MR at all but the lowest T _a fell to, or even below, the TNMR and the amplitude of the T _c rhythm increased from 2 to 5°C with increasing cold load. Under the most severe cold load, however, a further decrease of the minimum T _c was prevented by an increase of MR to 135% of the TNMR. Supplementing the continuously fed synthetic milk with noradrenaline (1.2 mg kg^–1 h^–1) during the minimum phase of the T _c rhythm increased MR upto 290% of the TNMR. The loose regulation of T _c during the minimum phase of the juvenile circadian T _c rhythm is thus not caused by a peripheral impairment of thermogenic capacitiy at the beginning of the light phase.     

Iron oxide/manganese oxide co-loaded hybrid nanogels as pH-responsive magnetic resonance contrast agents

This work described a proof of concept study of hybrid nanogel-based magnetic resonance contrast agents, SPIO@GCS/acryl/biotin@Mn-gel, abb. as SGM, for highly efficient, pH-responsive T₁ and T₂ dual-mode magnetic resonance imaging (MRI). SGM have been synthesized by assembling superparamagnetic iron oxide particles into polysaccharide nanoclusters, followed by in-situ reduction of the manganese species on the clusters and a final mild polymerization. The dual-mode SGM showed an interesting pH-responsiveness in in vitro MRI, with both T₁ and T₂ relaxivities turned “ON” in the acidic environment, along with an increase in the r₁ and r₂ relaxivity values by 1.7-fold (from 8.9 to 15.3 mM⁻¹ S⁻¹) and 4.9-fold (from 45.7 to 226 mM⁻¹ S⁻¹), due to desirable silencing and de-silencing effects. This interesting acidic-responsiveness was further verified in vivo with both significantly brightened signal of tumor tissue in T₁-weighted MR images and a darkened signal in T₂-weighted MR images 50 min post-injection of SGM. This smart hybrid nanogel may serve as a promising candidate for further studies of dual-mode (T₁ and T₂) contrast agents in MRI, due to its high stability, interesting pH-response mechanism and indicative imaging of tumors.     

Diagnosis of Pneumocystis pneumonia by real-time PCR in patients with various underlying diseases

BackgroundPneumocystis pneumonia (PCP) is a disease caused by the opportunistic infection of the fungus Pneumocystis jirovecii. Several PCR methods have been developed to aid in the diagnosis of PCP. In this study, we evaluated the performance of a real-time PCR in the diagnosis of PCP, in patients with various underlying diseases.MethodsNinety-seven BAL samples and 94 sputum samples from 191 patients were used in the study. Patients were classified as PCP (121 patients) or non-PCP (70 patients) based on their clinical and radiological presentations.ResultsReal time PCR amplified the P. jirovecii mitochondrial large-subunit rRNA gene with a detection limit of 68 copies of DNA per reaction. Non-PCP pathogens including 32 different fungi and bacteria were also evaluated. Overall, 71.9% of the samples from PCP patients and 14.5% of those from non-PCP patients were positive for the PCR test with a C_T value of the real-time PCR below 45. The main underlying diseases of the patients were hematological or solid malignancies (47.1%) and HIV infection (8.9%). The C_T values of the test were significantly lower in BAL samples from PCP patients than those from non-PCP patients (p = 0.024). No non-PCP patient had a C_T value below 30, whereas samples from 24.8% of PCP patients with underlying diseases had a C_T value below 30.ConclusionSince false positive PCR results were obtained, perhaps due to colonization, we suggest that the diagnosis of PCP should be based on a combination of clinical symptoms, underlying diseases, and PCR results.     

Does femoral strain distribution coincide with the occurrence of cervical versus trochanteric hip fractures? An experimental finite element study

The objective of this experimental finite element (FE) study is to test the hypothesis that strain distributions coincide with the occurrence of cervical versus trochanteric hip fractures during loading conditions simulating a sideways fall, and that the cervical versus trochanteric principal strain ratio predicts different fracture patterns. Cadaver femora (female, 83 ± 9 years) were CT scanned and mechanically tested simulating a fall. Thirteen cervical and 13 trochanteric fracture cases were selected for FE analysis. Principal strain distributions were analysed, and strain ratio ε_C/ε_T for strain patterns over the cervical and trochanteric regions was computed. The ratio ε_C/ε_T in the femora with cervical fractures (mean ± SD 1.103 ± 0.127) differed from that in trochanteric fractures (0.925 ± 0.137) (p = 0.001). The significant difference in the strain ratio between fracture types remained after accounting for femoral neck and trochanteric BMD (p = 0.014), showing that it is independent of BMD. Area under the ROC curve was 0.858 in the discrimination of fracture types. The model predicted the experimental fracture type correctly in 22 of 26 cases. The cervical versus trochanteric region principal strain ratio differed significantly between femora with experimental cervical versus trochanteric fractures, and 85% agreement was achieved for the occurrence of hip fracture types using a simple FE model.     

Severe Combined Immunodeficiency in Greek Children over a 20-Year Period

Severe combined immunodeficiencies (SCID) are a heterogeneous group of genetic disorders characterized by a blockade or impairment of both cellular and humoral immunity. Several epidemiological studies in different geographic areas have shown that the most common type of SCID affecting almost half of these patients is the X-linked common γ-chain (γ_c) deficiency. The objective of the study was to document the incidence and types of SCID in our area. We conducted a retrospective analysis of patients who were diagnosed with SCID in the major immunology center in Greece for a 20-year period. During the study period, 30 children from 27 unrelated families with final diagnosis of SCID were identified. The incidence of SCID in Greece is estimated at 1.7 cases per 100,000 live births. Out of 30 children, 19 were boys (63.3%) and 26 (86.7%) had Greek maternal origin. Lymphocyte immunophenotypes that were identified were T⁻B⁻NK⁺ in 12 (40%) children, T⁻B⁺NK⁻ in six (20%), T⁻B⁺NK⁺ in three (10%), T⁻B⁻NK⁻ in two (6.7%) and T⁺B^+/−NK⁺ in seven (23.4%) (among them, four [13.4%] females with Omenn’s syndrome). Molecular diagnosis was available for 12 children: γ_c (2) with non Greek maternal origin, Jak3 (2), Rag1 (2), Artemis (3), ADA deficiency (2), PNP deficiency (1). Out of the 26 children of Greek maternal origin diagnosed with SCID representing 23 distinct families, only two (8.7%) had lymphocyte immunophenotype compatible with γ_c-chain gene mutation (no molecular testing or enough DNA was available for them at the time of diagnosis). Findings of the present study suggest that, for unknown reasons, mutations of the γ_c chain of several cytokine receptors have a rare occurrence in our area.     

Thermodynamics of dimethylene urethane,of its ring‐opening polymerization,and of poly(dimethylene urethane) between 0 and 335 K

In an adiabatic vacuum calorimeter, the temperature dependence of the heat capacity C ⁰_p of dimethylene urethane (DMU) and poly(dimethylene urethane) (PDMU) was studied between 6 and 335 K with an uncertainty of about 0.2%. In a calorimeter with a static bomb and an isothermal shield, the energies of combustion ΔU_comb of the monomer and of the polymer were measured. From the experimental data, the thermodynamic functions C ⁰_p (T), H ⁰ (T)–H ⁰ (0), S ⁰ (T), G ⁰ (T)–H ⁰ (0) were calculated in the range from 0 to 335 K, and enthalpies of combustion ΔH ⁰_comb and thermochemical parameters of formation ΔH ⁰_f, ΔS ⁰_f, ΔG ⁰_f of the substances studied were estimated at T = 298.15 K and standard pressure. The results were used to calculate the thermodynamic characteristics of the ring‐opening polymerization of DMU in bulk (ΔH ⁰_pol, ΔS ⁰_pol, ΔG ⁰_pol) with formation of linear poly(dimethylene urethane) in the range from 0 to 330 K. In addition, the ceiling temperature T ⁰_ceil was determined.     

Evaluation of a Multitarget Real-Time PCR Assay for Detection of Bordetella Species during a Pertussis Outbreak in New Hampshire in 2011

A multitarget real-time PCR assay with three targets, including insertion sequence 481 (IS481), IS1001, and an IS1001-like element, as well as pertussis toxin subunit S1 (ptxS1), for the detection of Bordetella species was evaluated during a pertussis outbreak. The sensitivity and specificity were 77 and 88% (PCR) and 66 and 100% (culture), respectively. All patients with an IS481 C_T of <30 also tested positive by ptxS1 assay and were clinical pertussis cases. No patients with IS481 C_T values of ≥40 tested positive by culture. Therefore, we recommend that culture be performed only for specimens with IS481 C_T values of 30 ≤ C_T <40.     

Pseudomaximaler Nettoentzug von osmotisch freiem Wasser w?hrend der Harnkonzentrierung bei hypertoner Mannitdiurese

Zusammenfassung Während Hydropenie werden an kreislaufkompensierten Patienten mit unterschiedlich ausgeprägter essentieller Hypertonie und an nierengesunden normotensiven Kontrollen die Effekte, die intravenöse Infusion von 5% Kochsalzlösung bzw. von 10–12,5% Mannitlösung auf die Rückgewinnung von osmotisch freiem Wasser (T ^c h ₂ o) beim Harnkonzentrierungsvorgang ausüben, miteinander verglichen. Mit zunehmender Kochsalzdiurese strebt T ^c h ₂ o keinem Maximum zu, sondern steigt bei einer osmolalen Clearance (C _osm) von mehr als 20 ml/min und 1,73 m² Körperoberfläche progressiv mit C _osm über die bei vergleichbarer Mannitdiurese erzielten Maximalwerte an. Obgleich sich bei Patienten mit ausgeprägter essentieller Hypertonie funktionelle Störungen im Natriumstoffwechsel distaler Nephronabschnitte finden können, reagieren solche Probanden, solange die Nierenfunktion nicht nennenswert eingeschränkt ist, in dieser Beziehung qualitativ gleich wie gesunde Personen. Der progressive Anstieg von T ^c h ₂ o mit zunehmender Kochsalzdiurese ist auch bei Werten von C _osm, die 25 bis 30 ml/min überschreiten, nachweisbar. Hierbei kann während hypertoner Mannitdiurese bereits eine Tendenz zur Verminderung von T ^c h ₂ o auftreten. Bei nierengesunden normotensiven und leicht hypertensiven Personen erreicht T ^c h ₂ o Werte in der Größenordnung von 9 ml/min, wenn C _osm 30 ml/min, jeweils bezogen auf 1,73 m² Körperoberfläche, übersteigt.Einen eigentlichen Maximalwert für T ^c h ₂ o bzw. eine maximale Begrenzung des aktiven Natriumtransportes in das Nierenmark scheint es demnach beim gesunden Menschen und bei Patienten mit essentieller Hypertonie nicht zu geben. Es wird angenommen, daß es sich bei dem während hypertoner Mannitdiurese zu messenden Tm-Wert um ein Pseudomaximum handelt. Die Ergebnisse werden im Hinblick auf den Aussagewert von T ^c h ₂ o bei Störungen der Harnkonzentrierung diskutiert.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

A model-based analysis of pharmacokinetic–pharmacodynamic (PK/PD) indices of avibactam against Pseudomonas aeruginosa

ObjectiveThe aim of the present work was to use a semi-mechanistic pharmacokinetic–pharmacodynamic (PK/PD) model developed from in vitro time–kill measurements with P. aeruginosa to compare different pharmacodynamic indices derived from simulated human avibactam exposures, with respect to their degree of correlation with the modelled bacterial responses.MethodsA mathematical model of the effect of ceftazidime–avibactam on the growth dynamics of P. aeruginosa was used to simulate bacterial responses to modelled human exposures from fractionated avibactam dosing regimens with a fixed ceftazidime dosing regimen (2 or 8 g q8h as a 2-h infusion). The relatedness of the 24-h change in bacterial density and avibactam exposure parameters was evaluated to determine exposure parameter that closely correlated with bacterial growth/killing responses.ResultsFrequent dosing was associated with higher efficacy, resulting in a reduction of avibactam daily dose. The best-fit PD index of avibactam determined from the simulation was fT > C_T of 1 mg/L avibactam and q8h was the longest dosing interval able to achieve 2-log kill: 41–87% (3.3 h to 7.0 h out of 8-h interval, respectively). The avibactam exposure magnitude required to achieve a 2-log kill in the simulations was dependent on the susceptibility of the bacterial isolate to ceftazidime.ConclusionsAvibactam activity in combination with ceftazidime against multidrug resistant P. aeruginosa correlated with fT > C_T. Setting a threshold avibactam concentration to 1 mg/L, superimposed over a simulated human-like exposure of ceftazidime, achieved at least 2-log kill for the clinical dose of 500 mg q8h avibactam as a 2-h infusion, depending on the minimum inhibitory concentration of ceftazidime alone.     

The effect of the nonionic block copolymer pluronic P85 on gene expression in mouse muscle and antigen-presenting cells

DNA vaccines can be greatly improved by polymer agents that simultaneously increase transgene expression and activate immunity. We describe here Pluronic P85 (P85), a triblock copolymer of ethylene oxide (EO) and propylene oxide (PO) EO₂₆–PO₄₀–EO₂₆. Using a mouse model we demonstrate that co-administration of a bacterial plasmid DNA with P85 in a skeletal muscle greatly increases gene expression in the injection site and distant organs, especially the draining lymph nodes and spleen. The reporter expression colocalizes with the specific markers of myocytes and keratinocytes in the muscle, as well as dendritic cells (DCs) and macrophages in the muscle, lymph nodes and spleen. Furthermore, DNA/P85 and P85 alone increase the systemic expansion of CD11c⁺ (DC), and local expansion of CD11c⁺, CD14⁺ (macrophages) and CD49b⁺ (natural killer) cell populations. DNA/P85 (but not P85) also increases maturation of local DC (CD11c + CD86⁺, CD11c + CD80⁺, and CD11c + CD40⁺). We suggest that DNA/P85 promotes the activation and recruitment of the antigen-presenting cells, which further incorporate, express and carry the transgene to the immune system organs.     

Thermodynamics of 2‐Methyltrimethylene Urethane,its Polymerization in Bulk and of Poly(2‐methyl‐trimethylene urethane) Between 0 and 335 K

In an adiabatic vacuum calorimeter the temperature dependence of the heat capacity C ⁰_p of crystalline 2‐methyltrimethylene urethane (MTU; systematic name: 5‐methylperhydro‐1,3‐oxazin‐2‐one) and of amorphous poly(2‐methyltrimethylene urethane) [PMTU; systematic name: poly(5‐methyl‐2‐oxo‐1‐oxa‐3‐azahexamethylene)] was studied between 6 and 335 K with an uncertainty of about 0.2%. In a calorimeter with a static bomb and an isothermal shield the energies of combustion ΔU_comb of the monomer and of the polymer were measured. From the experimental data the thermodynamic functions C ⁰_p (T), H ⁰(T)–H ⁰(0), S ⁰(T), G ⁰(T)–H ⁰(0) were calculated in the range of 0 to 340 K, and enthalpies of combustion ΔH⁰_comb and thermodynamic parameters of formation ΔH ⁰_f, ΔS ⁰_f, ΔG ⁰_f of the substances studied were estimated at T = 298.15 K and standard pressure. The configurational entropy S ⁰_conf of the polymer was estimated. The results were used to calculate the thermodynamic characteristics of MTU polymerization in bulk (ΔH ⁰_pol, ΔS ⁰_pol, ΔG ⁰_pol) with ring‐opening and formation of linear PMTU in the range of 0 to 340 K.     

Impact of Long-Term Storage on Stability of Standard DNA for Nucleic Acid-Based Methods

Real-time PCR is dependent upon a calibration function for quantification. While long-term storage of standards saves cost and time, solutions of DNA are prone to degradation. We present here the benchmark treatment for preservation of DNA standards, involving storage in 50% glycerol-double-distilled water, whereby a deviation of 0.2 threshold cycle (C_T) values resulted after 100 days of storage.Detection of nucleic acid targets is an important tool in medical, biological, environmental, and food-related diagnostic applications (12). Among various methodical parameters, the issue of stable standards to ensure reliable quantifiable data is of prime importance. In real-time PCR, the threshold cycle (C_T) method is usually employed to obtain quantitative information based on comparison of the samples with external DNA standards. These DNA standards are measured, serially diluted, and then used for the calibration line. Storage of DNA standards would be desirable, as this production procedure is elaborate. In the past, many studies dealt with DNA degradation in the context of basic nucleic acid research, but few investigations covering practical applications in the context of molecular diagnostics were carried out (2, 5, 8, 20). Degradation of nucleic acids features in the storage of specimens, for example, cerebrospinal fluid and whole blood, and other samples, including various pathogens, and in forensic applications (1, 3, 6, 9, 10, 13, 16, 18, 21). In a pharmaceutical context, the stability of plasmids during storage has been investigated (7, 11, 15). All these studies have investigated storage by freezing based on rapid freeze and thaw cycles included in the experiments, but long-term storage has been neglected.The aim of the present study was to investigate solutions for standard DNA storage in the context of molecular diagnostics, including long-term storage in the experimental design.DNA from Listeria monocytogenes EGDe (ATCC-BAA-679) was used as the DNA quantification standard for real-time PCR. Bacteria were maintained at −80°C using the Microbank technology (Pro-Lab Diagnostics, Richmond Hill, Canada). All bacteria were grown overnight in tryptone soy broth with yeast extract (Oxoid, Hampshire, United Kingdom) at 37°C.For preparation of genomic DNA, 1 ml of a pure overnight culture was subjected to DNA isolation using the NucleoSpin tissue kit (Macherey-Nagel, Düren, Germany).Real-time PCR was carried out as described previously by targeting a 274-bp fragment of the prfA gene of L. monocytogenes (4, 17). Real-time PCR was performed in an Mx3000p thermocycler (Stratagene, La Jolla, CA), and the 25-μl volume contained either 5 μl of template DNA or, in the case of glycerol storage, 1 μl of template DNA and 4 μl of double-distilled water (ddH₂O).Genomic DNA samples were stored in 1× PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl; Invitrogen, Lofer, Austria); 50% glycerol (Merck, Darmstadt, Germany) in 1× PCR buffer; and 50% glycerol in ddH₂O. Aliquots of the different concentrations of DNA standards were stored for 100 days at 4, 0, and −20°C. Real-time PCR results for the standard dilutions containing 1.58 × 10⁵ and 1.58 × 10⁴ initial genomic Listeria DNA target copies have been integrated into high-copy-number standards (HCS). Standards containing 1.58 × 10³ and 1.58 × 10² initial target copies have been integrated into low-copy-number standards (LCS). During the 100-day period, measurements were carried out on a weekly basis by performing real-time PCR in duplicate. Changes in C_T values during the 100-day test interval were expressed as ΔC_T. The median (ΔC_T50) is the time-based average value at day 50. The final C_T value (ΔC_T100) is the average C_T value at the end of the test interval. The particular plots for all tested DNA standard concentrations and storage conditions are presented and itemized in the supplemental material with slopes and standard errors.After evaluation of the results for different temperatures, freezing at −20°C was found to provide the best storage conditions and caused the least shift in the resulting C_T values (ΔC_T) after real-time PCR. Nevertheless, this shift would lead to a significant deviation of the resulting sample values if such DNA standards were to be used as calibration standards. Storage in 50% glycerol and 1× PCR buffer also did not support durability of the genomic DNA. The results of these experiments for L. monocytogenes HCS (HCS_List) and LCS_List are presented in Table ​Table1.1. The mean standard errors for HCS_List and LCS_List under all tested conditions were 0.09 and 0.08 C_T values, respectively.

TABLE 1.

Results of long-term storage of L. monocytogenes genomic DNA